Genomic DNA extraction from whole blood

1. Genomic DNA extraction from whole blood

2. Principle • Genomic DNA Extraction Kit: In the high salt state, DNA purification resin adsorbed DNA specificly; while in a state of low-salt or aqueous solution, DNA was eluted down.

3. Reagents: • purification resin GN binding buffer washing buffer Purification Column ethanol

4. Steps： • 1. Add 0.4ml whole blood to 1ml purification resin in a tube. Invert tube 5-6 times gently and leave to incubate for 3 minutes at room temperature. Invert the tube again at the half of the 3 minutes. Spin at 5000 rpm for 3 secs. Discard supernatant. • 2. Re-suspend pellet in 1 ml of GN binding buffer, Invert the tube. Spin at 5000 rpm for 3 secs. Discard supernatant.

5. 3. Re-suspend pellet in 0.5 ml of washing buffer. Invert the tube. Spin at 5000 rpm for 3 secs. Discard supernatant. Repeat this step again. The pellet should be white to cream in colour. If pellet is significantly yellow, repeat washing step again.

6. 4. Add 0.8 ml of ethanol to pellet and re-suspend pellet. Transfer it to a new Purification Column. Spin at 12000 rpm for 1 min. Discard ethanol in the lower collection tube. Spin again, discard ethanol completely.

7. 5. Put the Purification Column in a new 1.5ml tube, add 100µl ddH2O to the Resin of the Purification Column, incubate for 3 minutes at room temperature, Spin at 12000 rpm for 2 min. • 6. Finally, the genomic DNA is in the tube.

8. Polymerase chain reaction

9. a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymaticreplication of the DNA.

10. A basic PCR set up requires several components and reagents.These components include: • DNA template that contains the DNA region (target) to be amplified. • Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. Taq PCR MasterMix • Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C. • Deoxynucleoside triphosphates(dNTPs), the building blocks from which the DNA polymerases synthesizes a new DNA strand. • Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. • Divalent cations; generally Mg2+ is used

11. CYCLES • Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA. • Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

12. CYCLES • Extension/elongation step: The temperature at this step depends on the DNA polymerase used; commonly a temperature of 72 °C is used with Taq polymerase. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction.

13. Final elongation: 70–74 °C for 5–15 minutes after the last cycle to ensure that any remaining single-stranded DNA is fully extended. • Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.

15. To get special gene from genomic DNA by PCR.

16. 1、Taq PCR MasterMix 2、primers 3、ddH2O 4、template DNA

17. ddH2O 21ul • 2X Taq PCR MasterMix 25ul • Primer 1 1ul • Primer 2 1ul • Template DNA 2ul 50ul

18. PCR Steps： lid 100℃ • Initialization step：94℃8min • 35 Cycles： Denaturation step 94℃30s Annealing step 55℃30s Extension step 72℃50s • Final elongation： 72℃ 10min • Final hold 4 ℃

19. NOTES • Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification.

20. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis.

21. The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (see Fig). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. • Initialization step: 94–96 °C for 1–9 minutes.

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